New molecular techniques for T. cruzi

摘要 克氏锥虫（Trypanosoma cruzi，T. cruzi）引起南美锥虫病（例如美洲锥虫病）， 人类这种感染是拉丁美洲的地方病;然而，由于从地方病的移民， 南美锥虫病在欧洲和美国都有发现。人惊讶的一点 克氏锥虫感染是随着疾病症状的发展而发展的 在急性感染后的几十年里对T.克鲁齐一直受到困难的限制， 基因操控我们使用了一种修饰的pTREX载体， 结构域（ddFKBP）来调节感兴趣的基因/蛋白。该载体系统允许快速和 在不同的T.克鲁齐生活报 循环阶段。利用这种技术，我们发现两种促分裂原活化蛋白激酶 TcMAPK 1和TcMAPK 3是T.克鲁兹我们计划开发一个条件 基于我们的ddFKBPpTREX载体系统的基因缺失系统。此外，快速过- 在ddFKBP系统中以调节方式表达致死基因应该是可行的， 这可以在多个T中完成。Cruzi分离物使用相同的载体构建体， 对分离株进行基因改造这种诱导致死表型T.克鲁兹会非常 可用于发病机理研究，允许在接种后的不同时间点消除微生物。 感染来剖析致病机制。这种寄生虫也可能代表 一种新的疫苗株，并将提供受控的免疫刺激。 因此，我们建议：（1）开发一个强大的条件敲除载体系统， TcMAPK 1和TcMAPK 3是T.基于ddFKBPpTREX的cruzi增长 向量。这一系统对其他必需基因的操纵也是有用的， 寄生虫;（2）我们还将创建T. cruzi允许受管制的 毒素基因的表达，当这些基因表达时将杀死这种寄生虫。这些 转基因寄生虫在确定病原体或作为疫苗株方面具有潜在的价值。

Abstract Trypanosoma cruzi (T. cruzi) causes Chagas Disease (e.g. American Trypanosomiasis) in humans. This infection is endemic to Latin America; however, due to immigration from endemic areas Chagas Disease is found in both Europe and the United States. What is striking about Infection with T cruzi is the development of chronic infection with disease symptoms manifesting decades after the acute infection. Research on T. cruzi has been limited by the difficulties in genetic manipulation. We used a modified pTREX vector with a ligand-controlled destabilization domain (ddFKBP) to regulate a gene/protein of interest. This vector system allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages. Using this technique, we found that two mitogen activated protein kinases (MAPK), TcMAPK1 and TcMAPK3, are essential for T. cruzi. We plan to develop a conditional gene deletion system based on our ddFKBPpTREX vector system. In addition, quickly over- expressing a lethal gene in a regulated fashion should be feasible in the ddFKBP system and this can be done in multiple T. cruzi isolates using the same vector construct without any need to genetically modify the isolates. Such an inducible lethal phenotype T. cruzi would be very useful for pathogenesis studies allowing elimination of the organism at various time points after infection to dissect the mechanisms of disease causation. Such parasites could also represent a new vaccine strain and would provide controlled immune stimulation. We, therefore, propose to: (1) develop a robust conditional knockout vector system using TcMAPK1 and TcMAPK3, essential genes for T. cruzi growth, based on our ddFKBPpTREX vectors. This system should be useful for the manipulation of other essential genes this parasite; and (2) we will also create vector systems for T. cruzi that allow the regulated expression of toxin genes that will kill this parasite when these genes are expressed. These transgenic parasites will be potentially valuable to define pathogeneses or as vaccine strains.